| OBJECTIVE:Automated immunohistochemical stainers available in the market cannot achieve superheating antigen retrieval at all as there are such problems as complex sample injection process,long staining time and worse staining result than manual ones.The objective of this study is to set up a new automatic immunohistochemical stainer to solve the problems mentioned above and ensure the reliability of immunohistochemical staining results achieved by automatic stainers.METHODS:Such critical steps as dewaxing,retrieval,cooling and antigen-antibody binding were improved to make the immunohistochemical staining process more suitable for automatic instruments and increase staining efficiency.In addition,immunohistochemical reactor that can perform superheating antigen retrieval and an array-type sample injection system were designed.The optimized staining process and the designed immunohistochemical reactor and sample injection system were applied to the new automatic immunohistochemical stainer.And we finished the construction and testing of the new instrument.RESULTS:Results of optimization on critical processes of immunohistochemical staining are as follows:1.Dewaxing process:replacement of xylene with a.new dewaxing agent does not affect the results of HE staining and immunohistochemical staining.The new dewaxing agent is nontoxic and nonvolatile;hence,it is more suitable for use in automated instruments.2.Retrieval process:for some antibodies such as ki-67 and ER,it is diff icult for non-pressurized EDTA or citrate antigen retrieval to achieve the staining effect achieved by superheating citrate antigen retrieval,though it is done by manual or by automated stainers that had been optimized.In addition,antibodies recommended for superheating citrate antigen retrieval account for 69.5%of commercial primary antibodies that need heat-mediated antigen retrieval.Therefore,the ultimate solution to this problem is to develop an automated immunohistochemical stainer that can conduct superheating antigen retrieval.3.Cooling process:the immunohistochemical staining effect of ki-67 by changing solution for cooling was the same as that by flushing the ektexine of reactor manually and the cooling time was reduced to 10min,only 1/3 of that of traditional instruments.Cooling by changing solution was easy to be controlled automatically and thus is more applicable to the automated immunohistochemical staining process.4.Antigen-antibody binding process:oscillation of slides made the incubation time of primary antibody reduce from 60min to 30min,which could become automatic easily.A new automated stainer was set up using the optimization mentioned above,as well as the improvements as follows:1.A reactor capable of resisting pressure of 0.1 MPa and conducting retrieval at 120°C was designed to realize superheating antigen retrieval.2.Modularized array-type sample injection system was designed to solve the problems of single sample injection heads,such as repeated washing,low sample injection efficiency and cross contamination among reagents.After test,the designed automated stainers realized superheating antigen retrieval and simplified the sample injection process.In addition,such autormated stainers could achieve the same staining result as the manual staining and were better than those available in the market;the completion time is only 63%of that of manual staining and 21%shorter than that of those stainers available in the market,which meant that staining efficiency were greatly improved.CONCLUSION:the problems of immunohistochemical stainers in the process of superheating antigen retrieval,cooling,sample injection,etc.were solved and the staining quality and efficiency were improved,which was of great significance in boosting automation and standardization of immunohistochemistry. |
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